Cacao (Theobroma cacao L.) is the second most important agricultural commodity in the international trade market for the tropical regions. Cacao powder and cacao butter, which are obtained from the processed cacao beans, are the most important ingredients in chocolate and confectionery products, and are also important additives in pharmaceutical and cosmetic products. Due to increasing demands for cacao-related products, there is an ever greater need for cacao trees with higher levels of productivity and improved cacao bean quality. Cacao trees have a high degree of genetic heterozygosity. A typical cacao planting contains a large population of trees grown from genetically different seeds, and relatively few trees produce exceptional yields, and about one third of the trees produce below average yields.
Since the majority of cacao commercially cultivated today is derived from a few varieties collected 50-60 years ago and has a narrow genetic base, cacao remains extremely vulnerable to diseases and other abiotic stresses. Up to 30% of the world cacao crop production is lost each year due to fungal and viral diseases and to attack by various insect pests (Wood and Lass 1987, Cocoa, 4th edition. Longman Sci & Tech and John Wiley & Sons, NY). In 1995, almost 50% of the total cacao bean production in Brazil was lost due to the witch's broom disease pathogen, and this raised concerns within the cacao industry, over the need for production of disease-resistant varieties. Continued improvement in cacao production, through the development and utilization of superior genotypes with desirable yield and bean quality characteristics, resistance to diseases and insect pests, and tolerance to drought and cold, via conventional breeding methods and biotechnology approaches, remains a great challenge. In this regard, the development of a reliable transformation system for cacao has become critical for the successful utilization of biotechnology for cacao tree improvement. Furthermore, it is desirable to be able to propagate vegetativelly higher-yielding trees to ensure uniform high yields.
During the past thirty years, attempts were made to use vegetative cloning of superior genotypes or selected trees produced through breeding as a means to increase the overall yield, quality, and agronomic performance of cacao. However, in spite of a great deal of effort over a number of years to devise improved methods for vegetative propagation, cacao trees are currently commercially reproduced only via cuttings. There are a number of disadvantages associated with the propagation of cacao plants via the rooting or grafting of plagiotropic cuttings. For example, this mode of propagation is expensive and labor intensive, propagation rates are low, there is a wide range of variation in the performance of individual cuttings, an undesirable bush-like growth pattern may occur, and there is a high degree of susceptibility to wind damage due to the lack of a taproot system. Thus, there is a great need in the art of cocoa cultivation for an efficient clonal propagation method that could provide plants agronomically similar to seed-derived plants.
Considerable effort was made to develop tissue culture-based propagation methods. However, cacao has proven to be notoriously recalcitrant to in vitro propagation (Flynn et al., (1990) Plant Tissue and Organ Cult. 20:111-117; Passey and Jones, (1983) J. Hort. Sci 58:589-592; Orchard et al., (1979) Physiol. Plant. 47:207-210).
Plant regeneration through somatic embryogenesis provides an alternative approach for clonal propagation of cacao. Somatic embryogenesis is the process by which somatic cells undergo bipolar development to give rise to whole plants by means of the development of adventitious embryos that occur without the fusion of gametes. Plants derived from somatic embryos are genetically identical to their parental donor cells, and have a taproot system and an orthotropic growth pattern similar to that of seed-derived plants.
Certain studies on somatic embryogenesis and plant regeneration of cacao have been performed. For example, Janick et al. (U.S. Pat. Nos. 4,204,366; 4,291,498; 4,301,619 and 4,545,147) and Sondahl et al. (U.S. Pat. No. 5,312,801) studied the possibility of inducing embryogenesis and regenerating plants of cacao.
Janick discloses a method for producing somatic embryos exclusively from immature zygotic embryo tissues of cacao using MS-based medium and increased (3-fold) CO.sub.2 concentration. In that procedure, the conversion or germination of somatic embryos into seedlings or plantlets was problematic and mature plantlets were not obtained. (See, e.g. Wang and Janick, (1984) Hort. Sci. 19:839-841). Furthermore, as pointed out by the same inventors in a subsequent publication (Figueira and Janick, (1993) Acta Hortic. 336:231-236), somatic embryos derived from immature zygotic embryos have limited value for commercial propagation, because cacao seeds are produced mainly through open pollination and the zygotic embryos used as a starting material are untested genotypes, i.e., the zygotic tissues are not genetically identical.
Sondahl developed a method for inducing somatic embryogenesis and plant production which uses a non-zygotic somatic tissues obtained from mature cacao plants as a starting material. The Sondahl procedure uses an MS-based culture medium and high sugar content. The procedure involves the following steps: (i) inducing a friable embryogenic callus from somatic tissues in a callus induction medium; (ii) recovering immature embryos from the friable embryogenic callus in a liquid culture medium; (iii) producing first stage somatic embryos in a regeneration medium; (iv) developing second stage somatic embryos in a differentiation medium with a high osmotic potential (80-120 g/l sucrose); and (v) germination of mature somatic embryos in plant regeneration medium. Sondhal uses ABA and GA hormones for embryo induction. In this procedure, regeneration of cacao plantlets depends primarily on the secondary somatic embryos induced from primary embryos subjected to an extended culture period. Up to 8 different types of culture media, and multiple growth regulators such as cytokinins (including zeatin, kinetin, 6-BA and 2-iP), auxins (including NAA and IAA), gibberellic acid, and abscisic acid were required.
The development of a procedure for inducing somatic embryogenesis in non-zygotic tissues as described by Sondahl did not eliminate the problem associated with in vitro propagation of cacao. The procedure could not be applied to all somatic tissues of cacao. Only two types of tissue explants, nucellus (the inner layer of an ovule) and young flower bud petals, were responsive to the established culture conditions and were capable of producing somatic embryos. This is a significant disadvantage since nucellus tissue can only be obtained from young cacao fruits and the availability of young fruits is often limited. Cacao plants generally have a low number of fruits because the majority of young fruits tend to abort during development.
More importantly, the Sondahl procedure resulted in a very low frequency of somatic embryogenesis and plant regeneration. For example, according to examples cited in the patent description, only 8 cacao plants were successfully established in the soil from 30,160 cultured nucellus explants that generated a total of 948 primary somatic embryo, and only 7 plants were produced from 27,721 cultured petal explants that produced a total of 167 primary embryos (U.S. Pat. No. 5,312,801). Additionally, the Sondahl procedure was tested using only two cacao genotypes (EET-162 and UF-667). A recent study by a French group using the Sondhal method demonstrated that only 5 among 25 tested cacao genotypes were capable of producing somatic embryos, while the rest remained non-responsive (Lopez-Baez et al., (1993) CRAS, Paris 316:579-584). Thus, the utilization of the procedures known in the art has never been attempted commercially. The low frequency of embryogenesis and plant production and the inability to produce somatic embryos from the majority of cacao genotypes have precluded the practical use of the Janick and Sondahl methods. Accordingly, there remains a need in the art for the development of an efficient method for the regeneration of cacao plants.
Applicants have now developed effective procedures for the stimulation of somatic embryogenesis and plant regeneration from somatic tissues of cacao. The present procedure has significant advantages over the Janick and Sondhal procedures. The procedure of the present invention uses novel culture media that are not based on the MS basal medium. In fact, Applicants have shown that the MS medium is toxic to cacao cells, which may explain the low efficiency of the Janick and Sondhal procedures. Furthermore, the method of the invention does not require high CO.sub.2 levels (as described by Janick) or high osmoticum (80-120 g/l sucrose as described by Sondhal). The embryo conversion medium of the invention is effective without high sucrose levels and without growth hormones. In contrast, the Sondhal procedure requires high osmoticum and hormones. Accordingly, Applicants have now surprisingly discovered a novel procedure and novel culture media that are more effective and simpler to use than those described by the prior art.
Genetic transformation of plant cells offers a unique method to modify the plant genetic milieu and thus expedite the introduction of valuable agronomic traits into existing genotypes. Two major approaches, biolistics (gene gun) and Agrobacterium tumefaciens-mediated gene transfer, have been developed for gene introduction in many plant species. The biolistics approach involves the introduction of DNA that is carried on metal particles which are accelerated by a high velocity force into target plant cells. Agrobactefium-mediated transformation is accomplished by utilizing the natural DNA delivery capabilities of the A. tumefaciens bacterium.
Over the years, attempts have been made by a number of research groups to develop workable transformation protocols for cacao using both of the above-mentioned methods. However, successful transformation of cacao using the biolistics approach has not been demonstrated. The reports of Purdy and Dickstein (Plant Disease 73: 638-639; 1989) and Sain et al. (Plant Cell Tiss Org Cult 37:243-251; 1994) provided the first evidence that a wild type A. tumefaciens strain is capable of transferring and integrating the T-DNA into the cacao genome. However, only non-regenerable tumorous callus tissue was obtained, and the use of non-tumorigenic strains of A. tumefaciens that had been modified to contain a disarmed Ti plasmid, failed to infect cacao cells.
Applicants have now developed a protocol for transformation of the somatic embryos and the production of transgenic cacao embryos and plants using non-tumorigenic strains of A. tumefaciens. Applicants are the first to obtain transformed cacao plants.